Abstract
Introduction
Intervertebral disc being an avascular tissue, the gap junction protein connexin play an important role in nutrient and extracellular matrix protein play a major role in its structural stability. The pathophysiology underlying the process of Disc Herniation is still not clear but genetic factors play a vital role in the early occurrence of the disease. The study was initiated to determine the association of Connexin 43 gene polymorphisms and protein levels in Disc herniated patients in comparison to healthy individuals in Indian population.
Methods
Pre Operative blood samples were collected from 20 patients undergoing surgery for disc herniation between the age group of 20–60years and 10 samples from healthy individuals with no history of Intervertebral disc disease. DNA was isolated, primers designed for exonic and UTR regions of Connexin 43 performed PCR and samples sequenced for analysis of polymorphic variants. Genotypic and phenotypic distributions were compared with Healthy controls. Hardy Weinburg calculations were done to correlate the allelic/genotypic frequencies in the both study groups. Levels of Connexin 43 were assayed by sandwich ELISA by utilizing a standard Streptavidin-HRP format.
Results
The sequencing of amplified PCR product of connexin 43 resulted in identification of 14 variants in the cytoplasmic region and 6 variants in the 3′UTR. The variant thr326pro, arg362 gln, arg366lys and arg376 gln were statistically significant. In the 3′ UTR there were 6 variants and almost all the herniated samples had 1 or 2 variants with C12508T statistically significant. There were multiple variants in individuals below the age of 40 years. Genetic variants both homozygous as well as heterozygous were more common in lower age groups of 20–40. The levels of connexin 43 were very high in herniated samples as compared with control (p < 0.0453).
Conclusion
Connexin43 polymorphisms at amino acid position 362 CGA-CAA (arginine to glutamine) and 376 CGG-CAG (arginine to gluramine) in herniated samples could hinder the hexamerisation of Connexon gap junction channel. The amino acid 362 and 376 are the binding sites for connexon hexamers after phosphorylation at the C- terminal region of the transmembrane protein. The functional connexin 43 is a transmembrane form. This shows that the 3′UTR polymorphism increases their expression but the protein does not get incorporated into the membrane due to polymorphic variants at the site of Arginine phosphorylation (position 362 and 376) and therefore is not physiologically functional. This profound reduction in gap junction channels could cause both nutrient and oxidative stress as the disc tissue is an avascular tissue the mode of channelizing between tissues is via the connexin gap junctions. The 3′UTR variants of connexin 43 can induce an increased expression of the mRNA. This study was further extended to check the levels of the protein in the disc lysates of herniated and control samples by ELISA. The Connexin 43 levels were very high in diseased samples (p < 0.0453) which can be hypothesized as biophysically nonfunctional gap junctional protein localized in the cytoplasma due to polymorphic variants at the site of Arg phosphorylation site which is one of the post translational requirements for hexamerisation of the connexin 43 and insertion into the membrane as channels. This is the first study to report a SNP associated mutation in gap junction protein Cx43.